Generation of quality-controlled SARS-CoV-2 variant stocks.

One of the main challenges in the fight against coronavirus disease 2019 (COVID-19) stems from the ongoing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into multiple variants. To address this hurdle, research groups around the world have independently developed protocols to isolate these variants from clinical samples. These isolates are then used in translational and basic research-for example, in vaccine development, drug screening or characterizing SARS-CoV-2 biology and pathogenesis. However, over the course of the COVID-19 pandemic, we have learned that the introduction of artefacts during both in vitro isolation and subsequent propagation to virus stocks can lessen the validity and reproducibility of data. We propose a rigorous pipeline for the generation of high-quality SARS-CoV-2 variant clonal isolates that minimizes the acquisition of mutations and introduces stringent controls to detect them. Overall, the process includes eight stages: (i) cell maintenance, (ii) isolation of SARS-CoV-2 from clinical specimens, (iii) determination of infectious virus titers by plaque assay, (iv) clonal isolation by plaque purification, (v) whole-virus-genome deep-sequencing, (vi and vii) amplification of selected virus clones to master and working stocks and (viii) sucrose purification. This comprehensive protocol will enable researchers to generate reliable SARS-CoV-2 variant inoculates for in vitro and in vivo experimentation and will facilitate comparisons and collaborative work. Quality-controlled working stocks for most applications can be generated from acquired biorepository virus within 1 month. An additional 5-8 d are required when virus is isolated from clinical swab material, and another 6-7 d is needed for sucrose-purifying the stocks.

A comparative study of in vitro air-liquid interface culture models of the human airway epithelium evaluating cellular heterogeneity and gene expression at single cell resolution.

BACKGROUND: The airway epithelium is composed of diverse cell types with specialized functions that mediate homeostasis and protect against respiratory pathogens. Human airway epithelial (HAE) cultures at air-liquid interface are a physiologically relevant in vitro model of this heterogeneous tissue and have enabled numerous studies of airway disease. HAE cultures are classically derived from primary epithelial cells, the relatively limited passage capacity of which can limit experimental methods and study designs. BCi-NS1.1, a previously described and widely used basal cell line engineered to express hTERT, exhibits extended passage lifespan while retaining the capacity for differentiation to HAE. However, gene expression and innate immune function in BCi-NS1.1-derived versus primary-derived HAE cultures have not been fully characterized. METHODS: BCi-NS1.1-derived HAE cultures (n = 3 independent differentiations) and primary-derived HAE cultures (n = 3 distinct donors) were characterized by immunofluorescence and single cell RNA-Seq (scRNA-Seq). Innate immune functions were evaluated in response to interferon stimulation and to infection with viral and bacterial respiratory pathogens. RESULTS: We confirm at high resolution that BCi-NS1.1- and primary-derived HAE cultures are largely similar in morphology, cell type composition, and overall gene expression patterns. While we observed cell-type specific expression differences of several interferon stimulated genes in BCi-NS1.1-derived HAE cultures, we did not observe significant differences in susceptibility to infection with influenza A virus and Staphylococcus aureus. CONCLUSIONS: Taken together, our results further support BCi-NS1.1-derived HAE cultures as a valuable tool for the study of airway infectious disease.

A neonatal mouse model characterizes transmissibility of SARS-CoV-2 variants and reveals a role for ORF8.

Small animal models have been a challenge for the study of SARS-CoV-2 transmission, with most investigators using golden hamsters or ferrets. Mice have the advantages of low cost, wide availability, less regulatory and husbandry challenges, and the existence of a versatile reagent and genetic toolbox. However, adult mice do not robustly transmit SARS-CoV-2. Here we establish a model based on neonatal mice that allows for transmission of clinical SARS-CoV-2 isolates. We characterize tropism, respiratory tract replication and transmission of ancestral WA-1 compared to variants Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Omicron BA.1 and Omicron BQ.1.1. We identify inter-variant differences in timing and magnitude of infectious particle shedding from index mice, both of which shape transmission to contact mice. Furthermore, we characterize two recombinant SARS-CoV-2 lacking either the ORF6 or ORF8 host antagonists. The removal of ORF8 shifts viral replication towards the lower respiratory tract, resulting in significantly delayed and reduced transmission in our model. Our results demonstrate the potential of our neonatal mouse model to characterize viral and host determinants of SARS-CoV-2 transmission, while revealing a role for an accessory protein in this context.

A neonatal mouse model characterizes transmissibility of SARS-CoV-2 variants and reveals a role for ORF8.

Small animal models have been a challenge for the study of SARS-CoV-2 transmission, with most investigators using golden hamsters or ferrets 1, 2 . Mice have the advantages of low cost, wide availability, less regulatory and husbandry challenges, and the existence of a versatile reagent and genetic toolbox. However, adult mice do not robustly transmit SARS-CoV-2 3 . Here we establish a model based on neonatal mice that allows for transmission of clinical SARS-CoV-2 isolates. We characterize tropism, respiratory tract replication and transmission of ancestral WA-1 compared to variants Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Omicron BA.1 and Omicron BQ.1.1. We identify inter-variant differences in timing and magnitude of infectious particle shedding from index mice, both of which shape transmission to contact mice. Furthermore, we characterize two recombinant SARS-CoV-2 lacking either the ORF6 or ORF8 host antagonists. The removal of ORF8 shifts viral replication towards the lower respiratory tract, resulting in significantly delayed and reduced transmission in our model. Our results demonstrate the potential of our neonatal mouse model to characterize viral and host determinants of SARS-CoV-2 transmission, while revealing for the first time a role for an accessory protein in this context.

In silico docking screen identifies airway host protease targets for human SERPINs.

Proteases play key roles in viral replication cycles. They can provide cleavage maturation of viral glycoproteins, processing of viral polyproteins, or disassembly of viral capsids. Thus, proteases constitute ideal targets for antiviral intervention â€" pharmaceutically, by small molecule inhibitors, or naturally, by host immune responses. Indeed, we and others have shown that individual members of the Serine protease inhibitor (SERPIN) family have specific antiviral function by blocking proteolytic steps inherent to viral replication cycles. Whether additional members of the large SERPIN family possess antiviral activity and whether SERPINs function as part of the antiviral cell-intrinsic immune response, is currently unknown. Here, we found that specific SERPINs are produced upon infection with clinically relevant respiratory viruses in vitro and in vivo , and in concert with classical interferon-stimulated genes. We next developed a structure-based in silico screen to uncover non-canonical SERPIN-protease pairs. We identified several SERPINs with potential antiviral function, including: SERPINE1 targeting cathepsin L, required for SARS-CoV-2 entry; SERPINB8 targeting furin, required for glycoprotein maturation cleavage of numerous viruses; and SERPINB2 targeting adenovirus protease, which suggests the first direct-acting antiviral SERPIN. Our study demonstrates how proteolysis is modulated for antiviral defense and how this process could inform antiviral targets against clinically relevant respiratory pathogens.

SARS-CoV-2 Portrayed against HIV: Contrary Viral Strategies in Similar Disguise.

SARS-CoV-2 and HIV are zoonotic viruses that rapidly reached pandemic scale, causing global losses and fear. The COVID-19 and AIDS pandemics ignited massive efforts worldwide to develop antiviral strategies and characterize viral architectures, biological and immunological properties, and clinical outcomes. Although both viruses have a comparable appearance as enveloped viruses with positive-stranded RNA and envelope spikes mediating cellular entry, the entry process, downstream biological and immunological pathways, clinical outcomes, and disease courses are strikingly different. This review provides a systemic comparison of both viruses' structural and functional characteristics, delineating their distinct strategies for efficient spread.

Viperin binds STING and enhances the type-I interferon response following dsDNA detection.

Viperin is an interferon-inducible protein that is pivotal for eliciting an effective immune response against an array of diverse viral pathogens. Here we describe a mechanism of viperin's broad antiviral activity by demonstrating the protein's ability to synergistically enhance the innate immune dsDNA signaling pathway to limit viral infection. Viperin co-localized with the key signaling molecules of the innate immune dsDNA sensing pathway, STING and TBK1; binding directly to STING and inducing enhanced K63-linked polyubiquitination of TBK1. Subsequent analysis identified viperin's necessity to bind the cytosolic iron-sulfur assembly component 2A, to prolong its enhancement of the type-I interferon response to aberrant dsDNA. Here we show that viperin facilitates the formation of a signaling enhanceosome, to coordinate efficient signal transduction following activation of the dsDNA signaling pathway, which results in an enhanced antiviral state. We also provide evidence for viperin's radical SAM enzymatic activity to self-limit its immunomodulatory functions. These data further define viperin's role as a positive regulator of innate immune signaling, offering a mechanism of viperin's broad antiviral capacity.

Tenth Scientific Biennial Meeting of the Australasian Virology Society-AVS10 2019.

The Australasian Virology Society (AVS) aims to promote, support and advocate for the discipline of virology in the Australasian region. The society was incorporated in 2011 after 10 years operating as the Australian Virology Group (AVG) founded in 2001, coinciding with the inaugural biennial scientific meeting. AVS conferences aim to provide a forum for the dissemination of all aspects of virology, foster collaboration, and encourage participation by students and post-doctoral researchers. The tenth Australasian Virology Society (AVS10) scientific meeting was held on 2-5 December 2019 in Queenstown, New Zealand. This report highlights the latest research presented at the meeting, which included cutting-edge virology presented by our international plenary speakers Ana Fernandez-Sesma and Benjamin tenOever, and keynote Richard Kuhn. AVS10 honoured female pioneers in Australian virology, Lorena Brown and Barbara Coulson. We report outcomes from the AVS10 career development session on "Successfully transitioning from post-doc to lab head", winners of best presentation awards, and the AVS gender equity policy, initiated in 2013. Plans for the 2021 meeting are underway which will celebrate the 20th anniversary of AVS where it all began, in Fraser Island, Queensland, Australia.

Lipid droplet density alters the early innate immune response to viral infection.

The cellular localisation of many innate signalling events following viral infection has yet to be elucidated, however there has been a few cases in which membranes of certain cellular organelles have acted as platforms to these events. Of these, lipid droplets (LDs) have recently been identified as signalling platforms for innate TLR7 and 9 signalling. Despite their wide range of similar roles in various metabolic pathways, LDs have been overlooked as potential platforms for antiviral innate signalling events. This study established an in vitro model to evaluate the efficiency of the early innate immune response in cells with reduced LD content to the viral mimics, dsDNA and dsRNA, and Sendai viral infection. Using RT-qPCR, the expression of IFN-ß and IFN-λ was quantified following stimulation along with the expression of specific ISGs. Luciferase based assays evaluated the combined expression of ISRE-promoter driven ISGs under IFN-ß stimulation. Cellular LD content did not alter the entry of fluorescently labelled viral mimics into cells, but significantly decreased the ability of both Huh-7 and HeLa cells to produce type I and III IFN, as well as downstream ISG expression, indicative of an impeded innate immune response. This observation was also seen during Sendai virus infection of HeLa cells, where both control and LD reduced cells replicated the virus to the same level, but a significantly impaired type I and III IFN response was observed in the LD reduced cells. In addition to altered IFN production, cells with reduced LD content exhibited decreased expression of specific antiviral ISGs: Viperin, IFIT-1 and OAS-1 under IFN-ß stimulation; However the overall induction of the ISRE-promoter was not effected. This study implicates a role for LDs in an efficient early innate host response to viral infection and future work will endeavour to determine the precise role these important organelles play in induction of an antiviral response.

Interferon-Stimulated Genes as Enhancers of Antiviral Innate Immune Signaling.

The ability of a host to curb a viral infection is heavily reliant on the effectiveness of an initial antiviral innate immune response, resulting in the upregulation of interferon (IFN) and, subsequently, IFN-stimulated genes (ISGs). ISGs serve to mount an antiviral state within a host cell, and although the specific antiviral function of a number of ISGs has been characterized, the function of many of these ISGs remains to be determined. Recent research has uncovered a novel role for a handful of ISGs, some of them directly induced by IFN regulatory factor 3 in the absence of IFN itself. These ISGs, most with potent antiviral activity, are also able to augment varying arms of the innate immune response to viral infection, thereby strengthening this response. This new understanding of the role of ISGs may, in turn, help the recent advancement of novel therapeutics aiming to augment innate signaling pathways in an attempt to control viral infection and pathogenesis.